The Wilms' tumor (wt1) gene encodes a protein referred to as WT1. As illustrated in FIG. 1 below, the WT1 protein is a 429 amino acid protein [SEQ ID NO:4] which contains four contiguous zinc fingers at the carboxyl-terminus, and a glutamine- and proline-rich region at the amino-terminus. Splice variants of WT1 can produce the protein with a 17 amino acid insert at amino acid 249 and/or a 3 amino acid insert at amino acid 390. The zinc finger region of WT1 has been shown to interact specifically with the early growth response (Egr)-1 DNA consensus sequence [see, e.g., Rauscher et al, Science, 250: 1259-1262 (1990); Wang et al, J. Biol. Chem., 267: 21999-22002 (1992); I. A. Drummond et al, Science, 257: 674-678 (1992); A. L. Gashler et al, Proc. Natl Acad. Sci., USA, 89: 10984-10988 (1992)]. The WT1 and Egr1 family of proteins are related by two criteria: they share approximately 50% homology within the zinc finger DNA binding domain, and they bind to the same nucleotide sequence of double stranded DNA using the zinc finger DNA binding domain. The zinc finger region of WT1 has been shown to interact specifically with other less defined sequences in the platelet derived growth factor (PDGF)-A chain promoter [Wang et al, J. Biol. Chem., 268: 9172-9175 (1993)].
WT1 is expressed in the nucleus of certain cells, and possesses the structural features of a DNA binding transcription factor. WT1 is a developmentally regulated transcription factor in the kidney which functions as a tumor suppressor. The amino-terminal region of WT1 protein mediates transcriptional suppression or activation in transient transfection assays [Madden et al, Science, 253: 1550-1553 (1991); Maheswaran et al, Proc. Natl. Acad. Sci. USA, 90: 5100-5104 (1993); S. L. Madden et al, Oncogene, 8: 1713-1720 (1993)].
The wt1 gene encoding WT1 protein is located on chromosome 11p13 and has been found to be mutated or deleted in a subset of hereditary and sporadic Wilms' tumors. Recently, high levels of wt1 expression were reported in tumors derived from these tissues such as ovarian carcinomas [Bruenig et al, Cancer Invest., 11: 393-399 (1993)], mesotheliomas [Park et al, cited above], and leukemias [Miwa et al, Leukemia, 7: 405-409 (1992), Miyagi et al, Leukemia, 7: 970-977 (1992)]. This is consistent with the notion that wt1 represents a tumor suppressor gene in this system [Haber et al, Cell, 61: 1257-1269 (1990)].
In contrast to some ubiquitously expressed tumor suppressor genes, such as the rb1 retinoblastoma susceptibility and the p53 gene, expression of wt1 mRNA appears to be developmentally restricted [Pritchard-Jones et al, Nature, 346: 194-197 (1990); Park et al, Nat. Genet., 4: 415 (1993)]. There are four different, e.g., alternatively spliced, WT1 mRNA messages produced by cells. WT1 mRNA expression is tightly regulated during differentiation of embryonic tissues and is present in background levels in only very specific organs and tissues in adults. For example, WT1 expression during embryonic development is detected in the kidney in the condensing metanephric mesenchyme and in the forming primitive renal vesicle, gonadal ridge mesothelia, and the mesothelial lining of the coelomic cavity and the organs that it contains. In addition, WT1 mRNA transcripts are detected in the spleen, the area posterior of the brain and the ventral horn motor neurons in the spinal cord. In the adult, normal levels of WT1 expression are found only in the kidney glomerular epithelial podocytes, the granulosa cells in the ovary follicles, and the sertoli cells of the testes.
Diagnostic methods for the ovarian carcinomas, mesotheliomas, and leukemias referred to above are based primarily on clinical attributes and histology of tumor specimens. These methods may at times not distinguish between closely related diseases and may lead to inappropriate treatments of patients. For example, in addition to the presence of many histological variants of malignant mesothelioma, there are other lesions that can affect the pleural surface and present a clinical and histological picture quite similar to malignant mesothelioma [R. J. Pisani et al, Mayo Clin. Proc., 63: 1234-1244 (1988)]. Additional relatively specific molecular markers that clearly distinguish between clinically similar lesions for malignant mesotheliomas as well as the other cancers would thus be a valuable clinico-pathological tool which will permit a precise diagnosis. This is important since treatment protocols and prognosis for such conditions vary significantly.
Currently available diagnostic tools include rabbit polyclonal antibodies for WT1 protein known in the art. Morris et al, Oncogene, 6: 2339-2348 (1991) describe two such antibodies which recognize amino acid fragments spanning amino acids 294-429 of SEQ ID NO:4 and amino acids 85-173 of SEQ ID NO:4, respectively, of the WT1 protein. Another rabbit polyclonal antibody, which recognizes WT1 amino acids 275-429 of SEQ ID NO: 4 was described by Telerman et al, Oncogene, 7: 2545-2548 (1992). Still other WT1 polyclonal antibodies are commercially available, e.g. the rabbit polyclonal antibody SC-192, which is available from Santa Cruz. However, while polyclonal antibodies in general are able to detect WT1 expression, they have disadvantages in their potential for cross-reactivity with closely related proteins which share common domains with the WT1 protein. These polyclonal antibodies by their nature are likely to provide inconsistent results in antigen specificity and binding affinity studies and are not particularly desirable for diagnostic uses.
Additionally, a commercially available mouse monoclonal antibody, DG-10 (Applied BioTechnology) was raised to the zinc finger region of WT1 and is known to cross-react with the Egr1 proteins. Expression of Egr1 proteins is not limited to cells or tissues that express WT1 and is independently regulated from WT1 expression. Therefore, any antibodies raised to the zinc finger domain in the carboxyl terminus of WT1 potentially may recognize Egr1 proteins.
Another anti-WT1 mouse monoclonal antibody has been described by Mundlos et al, Development, 119: 1329-1341 (1993). The Mundlos et al antibody is specific for a 17 amino acid sequence insert (See FIG. 1 below), i.e., a splice variant, that is present in only a subpopulation of the alternatively spliced WT1 mRNA messages. It is not anticipated to cross-react with Egr1.
Thus, there exists a need in the art for methods and compositions for detecting and differentially diagnosing conditions characterized by over-expression or inappropriate expression of WT1.